The present invention is generally related to the discovery of the therapeutic benefit of administering xcex3-tocopherol and xcex3-tocopherol derivatives. More specifically, the use of xcex3-tocopherol and racemic LLU-xcex1, (S)-LLU-xcex1, or other xcex3-tocopherol derivatives as antioxidants and nitrogen oxide scavengers which treat and prevent high blood pressure, thromboembolic disease, cardiovascular disease, cancer, natriuretic disease, the formation of neuropathological lesions, and a reduced immune system response are disclosed.
Vitamin E, an essential fat-soluble vitamin, encompasses eight naturally occurring compounds in two classes. The first class, tocopherols, have four members designated alpha, beta, gamma and delta. The two major forms, xcex1-tocopherol and xcex3-tocopherol, differ structurally only by a methyl group substitution at the 5-position. The second class, tocotrienols, are molecules related to the tocopherols and also consist of four members designated alpha, beta, gamma and delta. The tocotrienol structure differs from the tocopherols by possessing three double bonds in their side chain rather than being saturated.
One of the important chemical features of the tocopherols is that they are redox agents which act under certain circumstances as antioxidants. In acting as an antioxidant, tocopherols presumably prevent the formation of toxic oxidation products, such as perioxidation products formed from unsaturated fatty acids. Early on, investigators attributed most if not all of the biological activity of the tocopherols to their ability to act as antioxidants. More recently, however, other biological activities have been associated with tocopherols including the modulation of signal transduction, modulation of phospholipid metabolism, inhibition of protein kinase C, inhibition of phospholipase A and inhibition of prostaglandin production. (Meydani and Mosen, The Lancet 345(8943):170-175 (1995)).
Further, it has recently been discovered that individual members in the class of tocopherols may exhibit different biological properties from one another despite their structural similarity. Some investigators, for example, believe that xcex3-tocopherol, unlike xcex1-tocopherol, acts in vivo as a trap for membrane-soluble electrophilic nitrogen oxides and other electrophilic mutagens. (Christen et al. Proc. Natl. Acad. Sci. 94: 3217-3222 (1997)). In contrast, others report that xcex1-tocopherol is a more powerful antioxidant and has ten times the biological activity of xcex3-tocopherol. (Meydani and Mosen, The Lancet 345(8943):170-175 (1995)). Alpha-tocopherol is also thought to be retained in the body longer than xcex3-tocopherol and has been shown to preferentially reincorporate into nascent very low-density lipoproteins (LDL). (Christen et al. Proc. Natl. Acad. Sci. 94: 3217-3222 (1997)). At present, an understanding of the differences in biological activity of the four tocopherols and their effect on the body is in its infancy.
Alpha tocopherol is largely considered the most important member of the class of tocopherols because it constitutes about 90% of the tocopherols found in animal tissues and displays the greatest biological activity in the commonly used bioassay systems. In consequence, vitamin E supplements are almost exclusively made of xcex1-tocopherol and little investigation into the efficacy of supplementation with xcex3-tocopherol has been conducted.
The therapeutic benefits of vitamin E supplementation remains a subject of considerable debate. Several studies have proposed that vitamin E supplementation may prevent a plethora of ills but many of these studies fail to provide causal connections between vitamer supplementation and therapeutic benefit; they merely indicate that a high dietary or plasma concentration and supplemental intake of vitamin E is associated with a reduced risk of disease. In fact, some studies have failed to demonstrate that tocopherol supplementation provides any protection from disease. (Meydani and Mosen, The Lancet 345(8943):170-175 (1995) and (Christen et al. Proc. Natl. Acad. Sci. 94: 3217-3222 (1997)). A reliable method to treat and prevent diseases associated with oxidative stress and vitamin E deficiency is highly desirable.
The present invention reveals the discovery of the therapeutic benefit of administering xcex3-tocopherol and xcex3-tocopherol derivatives such as LLU-xcex1. The novel use of xcex3-tocopherol and xcex3-tocopherol derivatives as antioxidants and nitrogen oxide scavengers which treat and prevent high blood pressure, thromboembolic disease, cardiovascular disease, cancer, natriuretic disease, the formation of neuropathological lesions, and a reduced immune system response are disclosed.
One embodiment of the present invention is a medicament comprising xcex3-tocopherol and LLU-xcex1 with and without additional active ingredients that are effective in producing a natriuretic effect. Another embodiment is a medicament comprising xcex3-tocopherol, xcex1-tocopherol, and LLU-xcex1 with and without additional active ingredients that are effective in producing a natriuretic effect. A further embodiment is a medicament comprising xcex3-tocopherol, xcex2-tocopherol, and LLU-xcex1, with and without additional active ingredients that are effective in producing a natriuretic effect. Still further, an embodiment comprising xcex1-tocopherol, xcex3-tocopherol, xcex2-tocopherol, and LLU-xcex1, with and without additional active ingredients that are effective in producing a natriuretic effect, is disclosed. In the alternative, the embodiments described above may include (S)-LLU-xcex1 or other xcex3-tocopherol derivatives instead of LLU-xcex1.
According to the methods of treatment and prevention disclosed, the medicaments described above are administered to subjects suffering from high blood pressure, thromboembolic disease, atherosclerosis, cardiovascular disease, cancer, natriuretic disease, the formation of neuropathological lesions, and a reduced immune system response. One method involves the administration of a therapeutically beneficial amount of xcex3-tocopherol, with or without supplementation of LLU-xcex1, to subjects suffering from a high blood pressure so as to treat and prevent this condition. By another method, a therapeutically beneficial amount of xcex3-tocopherol, with or without supplementation of LLU-xcex1, is administered to treat and prevent thromboembolic disease. A related method to treat and prevent the aggregation of platelets and/or binding of platelets to adhesive proteins is also disclosed.
Another method contemplated by the present inventor involves the administration of a therapeutically beneficial amount of xcex3-tocopherol, with or without supplementation of LLU-xcex1, to treat and prevent cardiovascular diseases, such as ischemia, angina, edematous conditions, artherosclerosis, LDL oxidation, adhesion of monocytes to endothelial cells, foam-cell formation, fatty-streak development, platelet adherence, platelet aggregation, smooth muscle cell proliferation, and reperfusion injury. Further, a method to treat and prevent cancers, such as lung cancer, prostate cancer, breast cancer, and colon cancer by administering a therapeutically beneficial amount of xcex3-tocopherol, with or without supplementation of LLU-xcex1 are presented.
Methods of treatment and prevention of natriuretic diseases, such as hypertension, high blood pressure, ischemia, angina pectoris, congestive heart failure, cirrhosis of the liver, nephrotic syndrome, ineffective renal perfusion, or ineffective glomerular filtration, by administering a therapeutically beneficial amount of xcex3-tocopherol, with or without supplementation of LLU-xcex1 are also provided. Additionally, methods of treating and preventing neurological diseases including hyporeflexia, opthalmoplegia, and axonal dystrophy using a therapeutically beneficial amount of xcex3-tocopherol, with or without supplementation of LLU-xcex1, are described. Finally, methods to improve a subject""s immune system response and a related method to reduce the production of free-radicals by administering a therapeutically beneficial amount of xcex3-tocopherol, with or without supplementation of LLU-xcex1, is revealed.
FORMULA A shows the structural formula of LLU-xcex1. 
FORMULA B shows the structural formula of (S)-LLU-xcex1. 
In accordance with the present invention, a novel method for the treatment and prevention of high blood pressure, thromboembolic disease, atherosclerosis, cancer, natriuretic disease, the formation of neuropathological lesions, and a reduced immune system response is provided. The method involves administering orally or parenterally substantially pure xcex3-tocopherol or a formulation comprising xcex3-tocopherol and racemic LLU-xcex1, (S)-LLU-xcex1, or other xcex3-tocopherol derivative.
By xe2x80x9cLLU-xcex1xe2x80x9d is meant the compound 6-hydroxy-2,7,8-trimethylchroman-2-propanoic acid, molecular weight of 264.1362 and molecular formula of C15H20O4. LLU-xcex1 may be in the racemic form or as the S enatiomer (also denoted as (S)-LLU-xcex1). A general discussion of the isolation and characterization of LLU-xcex1 is provided by Wechter et al. (U.S. patent application Ser. No. 08/290,430) the disclosure of which is hereby incorporated by reference.
By xe2x80x9cxcex3-tocopherol derivativexe2x80x9d is meant xcex3-tocopherol metabolites and synthetic chroman derivatives including, but not limited to, LLU-xcex1, LLU-xcex3, racemic chromans, chroman methyl esters, chroman esters, chroman amides, R4 chroman esters, oxidized chroman derivatives, racemic 2,5,7,8-tetramethyl-2-(xcex2-carboxyethyl)-6-hydroxy chroman, 2,5,7,8-tetramethyl-2-(xcex2-carboxyethyl)-chroman, 2,7,8-trimethyl-2-(xcex2-carboxyethyl) chroman, racemic 4-methyl-6-(5,6-dimethylbenzohinoyl)-4-hexanolid, 4-Methyl-6-(3,5,6-trimethylbenzochinoyl)-4-hexanolid, (S)-4-Methyl-6-(5,6-dimethylbenzochinoyl)-4-hexanolid, 2,7,8-Trimethyl-2-(xcex2-carboxyethyl)-6-acetyl chroman, 2,7,8-Trimethyl-2-(xcex2-carboxyethyl)-6-acetyl chroman methyl ester, and benzodipyran methyl ester. Many xcex3-tocopherol derivatives are natriuretic compounds but the meanining of xe2x80x9cxcex3-tocopherol derivativexe2x80x9d is not intended to be limited to only natriuretic compounds. Other xcex3-tocopherol metabolites and synthetic chroman derivatives may be known by those of skill in the art or will be discovered in the future and are encompassed by this definition.
By xe2x80x9cnatriuretic diseasexe2x80x9d is meant diseases associated with abnormal excretion of sodium from the body. The term natriuretic disease includes but is not limited to hypertension, high blood pressure, ischemia, angina pectoris, congestive heart failure, cirrhosis of the liver, nephrotic syndrome, ineffective renal perfusion, and ineffective glomerular filtration, or any combination thereof. Other forms of natriuretic disease will be apparent to those of skill in the art and are encompassed by the definition as used in this invention.
As used herein, the term xe2x80x9cnatriuretic compoundxe2x80x9d refers to a compound which increases the rate of sodium excretion without contributing to significant potassium loss in a mammal upon administering the compound to the mammal. The term xe2x80x9cnatriuretic compoundxe2x80x9d also refers to both the native compound and in vitro or in vivo modifications which retain natriuretic activity. It is understood that limited modifications, substitution or deletions of functional groups may be made without destroying the biological activity. Moreover, it will be recognized by those skilled in the arts of chemistry and pharmaceutical preparation that many derivatives can be made which are biologically and chemically equivalent to, or even more active than, the indicated compounds hereinafter. Examples of equivalent compounds include esters, ethers, amides and salts of the foregoing compounds.
xe2x80x9cSubstantially purified,xe2x80x9d when used to describe the state of the natriuretic compound, denotes the compounds essentially free of proteins, steroids, and other material normally associated or occurring with natriuretic compounds in its native environment.
As used herein, the term xe2x80x9cpost salt peakxe2x80x9d refers to material eluted from a G-25 Sephadex column which appears immediately after the sodium, potassium, urea and creatinine containing fractions which has uv. absorbance at 290 nm.
A material is xe2x80x9cbiologically activexe2x80x9d if it is capable of increasing natriuresis in an in vivo assay as described herein.
By xe2x80x9cthromboembolic diseasexe2x80x9d is meant diseases characterized by platelet aggregation, platelet adhesion to adhesive proteins, or platelet hyperactivity. Although thromboembolic disease is commonly associated in insulin-dependent diabetic patients, this understanding is not intended to limit the invention. Elderly patients and patients suffering from various forms of cardiovascular disease exhibit platelet aggregation, platelet adhesion to adhesive proteins, and platelet hyperactivity which can be defined as forms of thromboembolic disease for the purposes of this invention. Other forms of thromboembolic disease will be apparent to those of skill in the art and are encompassed by the definition used in this invention.
By xe2x80x9ccardiovascular diseasexe2x80x9d is meant diseases associated with the cardiopulmonary and circulatory systems including but not limited to ischemia, angina, edematous conditions, artherosclerosis, LDL oxidation, adhesion of monocytes to endothelial cells, foam-cell formation, fatty-streak development, platelet adherence, and aggregation, smooth muscle cell proliferation, reperfusion injury, and other conditions known by those of skill in the art to be related to the pathogenesis of cardiovascular disease.
By xe2x80x9ccancerxe2x80x9d is meant diseases that have been associated with mutagenesis, cell transformation, oncogenesis, neoplasia, or metastasis, including but not limited to, various forms of lung cancer, prostate cancer, breast cancer, and colon cancer, or any combination thereof. Other forms of cancer will be apparent to those of skill in the art and are encompassed by the definition used in this invention.
By xe2x80x9cneurological diseasexe2x80x9d is meant diseases associated with the brain and nervous system, including but not limited to, hyporeflexia, proprioception, opthalmoplegia, and axonal dystrophy. Other forms of neurological diseases will be apparent to those of skill in the art and are encompassed by the definition as used in this invention.
Gamma-tocopherol is a water-insoluble, non swelling amphiphile, as are triglycerides and cholesterol. Thus, many of the processes involved in the absorption of lipids are also required for absorption of xcex3-tocopherol such as emulsification, solubilization within mixed bile salt micelles, uptake by the small intestine, packaging within lipoprotein particles, and secretion into the circulation via the lymphatic system. Gamma-tocopherol is transferred to tissues in much the same manner as other lipids and spontaneous transfer and exchange of tocopherol between cell membranes has been documented. Since xcex3-tocopherol is rapidly absorbed in the lipids of various tissues including the liver, its antioxidant and radical scavenger activities primarily occur in the lipid phase and only tangentially in the aqueous phase. LLU-xcex1, on the other hand, is considerably more hydrophilic than xcex3-tocopherol and acts as an antioxidant, a natriuretic compound, and radical scavenger in primarily the aqueous phase. Thus, the present inventor contemplates a method to treat and prevent disease which employs supplements comprising xcex3-tocopherol with and without fortification with racemic LLU-xcex1, (S)-LLU-xcex1, or other xcex3-tocopherol derivative so as to selectively provide natriuretic redox agents to the lipid and aqueous phases of a patient""s body.
The preparation of soft gelatin capsules comprising commercially available xcex3-tocopherol in doses of 200 to 800 mg is understood by those of skill in the art. The xcex3-tocopherol may be present as the free alcohol or the acetate or succinate ester. A supplement of xcex3-tocopherol preferably contains at least 60-65% (weight to weight) xcex3-tocopherol and up to 10% xcex1-tocopherol and 25% xcex2-tocopherol as isolated from soy oil, or in certain circumstances up to 25% xcex4-tocopherol. Particularly preferred compositions include at least 70% xcex3-tocopherol. These formulations are only intended to guide one of skill in the art and formulations of xcex3-tocopherol that would be effective for use in the disclosed methods may include as low as 50% (weight to weight) xcex3-tocopherol or up to 100% (weight to weight) xcex3-tocopherol, but desirably contain 55% (weight to weight) xcex3-tocopherol to 95% (weight to weight) xcex3-tocopherol.
In another embodiment of this invention, soft gelatin capsules comprising commercially available xcex3-tocopherol are fortified with a natriuretic compound such as LLU-xcex1, (S) LLU-xcex1, or other xcex3-tocopherol derivative some of which may be present as the free acid or a simple ester. One aspect of the invention, for example, comprises a natriuretic compound having the formula I: 
in which
R is O, S, SO, SO2, a secondary or tertiary amine group, a phosphate group, a phosphoester group, or an unsubstituted or substituted methylene group,
R1 and R2 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic or aromatic ring,
R3 and R4 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic, aromatic or heterocyclic ring,
R5 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester or unsubstituted or substituted amine,
R6 is COOH, COOR7, CONH2, CONHR7, CONR7R8, NH2, NHR7, NR7R8, or a carboxylate salt,
R7 and R8 independently are unsubstituted or substituted alkyl, aryl, alkaryl, aralkyl, alkenyl or alkynyl,
n is 0 to 3, and
m is 0 to 5.
As used herein, the term xe2x80x9csubstitutedxe2x80x9d denotes the presence of one or more substituent such as alkyl, aryl, alkaryl, aralkyl, ether or halogen. More particular substituents include C1-6 unbranched or branched alkyl, such as methyl, ethyl, propyl, n-butyl, sec-butyl and tert-butyl, and C6-12 aryl, particularly phenyl.
In a preferred embodiment, R is O. Also preferably, n=1. Preferably, m=2.
R6 preferably is COOH.
Preferably, R3 is H or OH. Also preferably, R4 is H or CH3.
In a preferred embodiment, R1, R2 and R5 are CH3.
Exemplary preferred compounds of formula I include those in which R is O, R1, R2 and R5 are CH3, R3 is OH, R4 is H or CH3, R6 is COOH, n=1 and m=2.
Other exemplary preferred compounds of formula I includes those in which R is O, R1, R2 and R5 are CH3, R3 is H, R4 is H or CH3, R6 is COOH, n=1 and m=2.
In a preferred embodiment, R7 is a C1-6 alkyl group, in particular CH3.
In another preferred embodiment, R3 is OH.
Compounds used in the present invention can also be obtained by modifying the above recited formula in numerous ways while preserving natriuretic activity. Examples of such active derivatives include compounds of formulae II-V, below.
In all formulae described herein, moieties having like designations are considered to correspond to each other as like moieties in related compounds.
Another aspect of the invention comprises natriuretic compounds having the formula II: 
wherein
R1 and R2 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic or aromatic ring,
R3 and R4 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic, aromatic or heterocyclic ring,
R5 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester or unsubstituted or substituted amine,
R6 is COOH, COOR7, CONH2, CONHR7, CONR7R8, NH2, NHR7, NR7R8, or a carboxylate salt,
R7 and R8 independently are unsubstituted or substituted alkyl, aryl, alkaryl, aralkyl, alkenyl or alkynyl,
R9 is hydroxyl or unsubstituted or substituted alkoxyl,
n is 0 to 3, and
m is 0 to 5.
In a preferred embodiment, R1, R2 and R5 are CH3. Preferably, R3 is OH.
R4 preferably is H.
Additionally, it is preferred that n=1. Preferably m=2.
In a preferred embodiment, R6 is COOCH2CH3 and r9 is OH. In another preferred embodiment, R6 is COOH and R9 is CH3CH2O.
Specific examples includes the following: 
A further aspect of the invention comprises natriuretic compounds having the formula III: 
wherein
R1 and R2 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic or aromatic ring,
R3 and R4 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic, aromatic or heterocyclic ring,
R5 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester or unsubstituted or substituted amine,
n is 0 to 3, and
q is 0 to 4.
In preferred embodiments, n=1. Also preferred are compounds in which m=2.
Exemplary natriuretic compounds of formula III include the following: 
The instant invention comprises other natriuretic compounds having the formula IV: 
wherein
R1 and R2 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic or aromatic ring,
R4 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl,
R5 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester or unsubstituted or substituted amine,
R6 is COOH, COOR7, CONH2, CONHR7, CONR7R8, NH2, NHR7, NR7R8, or a carboxylate salt,
R7 and R8 independently are unsubstituted or substituted alkyl, aryl, alkaryl, aralkyl, alkenyl or alkynyl,
R9 is hydroxyl or unsubstituted or substituted alkoxyl,
n is 0 to 3, and
m is 0 to 5.
Preferably n=1. Also, preferably m=2.
Specific compounds of the invention according to formula IV include: 
Natriuretic compounds of formula V are also combined with xcex3-tocopherol to make the medicaments of the instant invention: 
wherein
R1 and R2 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic or aromatic ring,
R4 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl,
R5 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester or unsubstituted or substituted amine,
n is 0 to 3, and
q is 0 to 4.
Preferred embodiments are those in which n=1. Also, it is preferred that m=2.
Included in the inventive compounds of formula V are: 
In accordance with another aspect of present invention, medicaments having the formula Ia and xcex3-tocopherol are contemplated. 
in which
R is O, S, SO, SO2, a secondary or tertiary amine group, a phosphate group, a phosphoester group, or an unsubstituted or substituted methylene group,
R1 and R2 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic or aromatic ring,
R3 and R4 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic, aromatic or heterocyclic ring,
R5 is H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ester or unsubstituted or substituted amine,
R6 is COOH, COOR7, CONH2, CONHR7, CONR7R8, NH2, NHR7, NR7R8, or a carboxylate salt,
R7 and R8 independently are unsubstituted or substituted alkyl, aryl, alkaryl, aralkyl, alkenyl or alkynyl,
n is 0 to 3, and
m is 0 to 5.
In preferred embodiments,
(i) when R is O, R1, R2 and R5 are CH3, R3 and R6 are OH, and R4 is H, m=2 to 5;
(ii) when R is O R1 is H or CH3, R2 is H, CH3, C(CH3)3 or CH(CH3)2, R3 is OH or CH3COO, R4 is CH3 or CH(CH3)2, R is H, CH3or CH2CH3, and R6 is H, OH, OCH3, OCH2CH3 or NH2, m=1 to 5;
(iii) when R is O, R1 and R5 are CH3, R2 and R4 are H, R3 is OH or CH3COO, and R6 is OH or CH3O, m is not 2;
(iv) when R is O, R1, R2 and R5 are CH3, R3 is OH or CH3COO, R4 is alkyl having at least two carbon atoms, and R6 is H, OH or ester, m=1; and
(v) when R1, R2 and R5 are methyl, R3 and R6 are OH and R4 is alkyl, m=2.
Certain medicaments of the present invention comprise natriuretic compounds that have been isolated in substantially pure form. The natriuretic compounds are obtained from a variety of sources, including urine, hypothalamus, adrenal, liver, kidney, plasma, blood and cultured cells. Human uremic urine is the preferred source, although normal human urine or hypertensive human urine may also be used.
One of the isolated natriuretic compounds used to make a medicament of the present invention is LLU-xcex1. (See FIGS. 1 and 2). LLU-xcex1 has the following properties: a major ultraviolet absorbance peak at about 210 nm; a broad secondary peak at about 295 nm; instability in dilute base; capability of esterification by reaction with CH2N2. The compound is capable of increasing sodium excretion in the urine in mammals without a corresponding increase in potassium excretion, and does not cause a significant change in mean arterial pressure. The compound additionally acts as a cardio-selective free radical scavenger.
Medicaments of the instant invention also comprise another isolated natriuretic compound, named LLU-xcex3, which has the following properties: a major ultraviolet absorbance peak at about 220 nm; a secondary peak at about 268 nm; high instability in the presence of O2 or in dilute base. It is capable of increasing sodium excretion in mammalian urine without a corresponding increase in potassium excretion, although potassium excretion (kaliuresis) may be observed occasionally after infusion of the compound into conscious rats. In addition, it does not cause a significant change in mean arterial pressure and it shows no inhibition of the sodium pump.
Natriuretic compounds which comprise the present invention can be purified by a number of methods, particularly those exemplified herein. In a preferred method within the invention, collected urine is processed by ultrafiltration (xe2x89xa63 kDa), gel filtration chromatography (G-25) and extraction with isopropanol and diethyl ether. The organic soluble material is then subjected to sequential high-performance liquid chromatography, while assaying for the natriuretic, activity in vivo. Alternatively, collected urine is extracted with ether, separated by high performance liquid chromatography, and fractions are assayed for natriuretic activity.
In a further alternative embodiment, the natriuretic compounds in the medicaments of the present invention can be synthesized using methods known to those skilled in the art. One such method is the method described by J. Weichet et al., Czech. Chem. Commun. 24, 1689-1694 (1959), the disclosure of which is hereby incorporated by reference. This method can readily be adapted by one of ordinary skill in the art to provide a method of synthesizing the compounds of the present invention. Other methods to synthesize the natriuretic compounds of the present invention are disclosed in Wechter et al., Proc. Natl. Acad. Sci. USA 93:6002-6007 (1996) and Kantoci et al., J. Pharmacology and Experimental Therapeutics 282:648-656 (1997) which are hereby incorporated by reference.
A preferred synthetic method includes the step of reacting a compound of the formula VI: 
in which
R1 and R2 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl. or jointly complete a 5- or 6-member aliphatic or aromatic ring, and
R3 and R4 independently are H, OH, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen or unsubstituted or substituted sulfonyl, or jointly complete a 5- or 6-member aliphatic, aromatic or heterocyclic ring,
with a vinyl lactone of the formula VII: 
in which
R5 is H, alkyl, aryl, alkenyl, alkynyl, aromatic or ester,
n is 0 to 3, and
q is 0 to 4.
In a preferred embodiment of the foregoing synthesis, R3 is OH. Preferably, R4 is not simultaneously OH. A preferred compound of formula VI is a hydroquinone, for example 2,3-dimethyl-1,4-hydroquinone.
A preferred vinyl lactone of formula VII is xcex3-methyl-xcex3-vinylbutyrolactone (R5=CH3, n=1, q=1).
In carrying out the foregoing reaction, preferably a catalyst is used, such as a metallic or non-metallic salt. Specific types of catalyst include non-metallic salts which form complexes with a solvent, particularly a catalyst such as boron trifluoride diethyl etherate.
In carrying out the foregoing reaction, preferably an aprotic or protic solvent is employed, in particular an aprotic solvent such as dioxane. The catalyst and/or the vinyl lactone is preferably diluted in the selected solvent.
Preferably the synthesis is carried out at an elevated temperature, such as 100-110xc2x0 C.
In a preferred embodiment, the foregoing reaction mixture is diluted with an aprotic or protic solvent, particularly an aprotic solvent such as diethyl ether.
The desired product preferably is obtained from concentrated supernatant which is purified, for example, using an RP-HPLC column or silica gel. Preferred eluents for RP-HPLC include mixtures of water, acetonitrile and acetic acid. Preferred solvents for silica gel include ethyl acetate and hexane. Other purification methods, such as crystallization, can be used. Also, other eluents, such as hexane and dimethyl ketone, can be employed.
The foregoing synthesis produces a racemic mixture, of which typically one enantiomer is active while the second enantiomer is less active or inactive. The racemate can be employed in compositions according to the invention, with adjustment of the quantity to account for the presence of the inactive enantiomer. Alternatively, the racemate can be resolved using conventional methods, and the active enantiomer identified and utilized. All enantiomeric forms of the compounds described herein are specifically contemplated as being within the scope of the instant invention.
As a byproduct of the foregoing synthesis, derivative compounds of formula VIII are produced: 
These compounds can also be employed as natriuretic compounds which comprise the medicamnet according to the instant invention. Exemplary compounds of formula VIII include the following benzodipyran derivatives: 
All stereoisomeric forms of the foregoing compounds, including meso compounds and diastereomeric pairs, are specifically contemplated as being within the scope of the instant invention.
Di-oxidized and/or di-hydrated derivatives of the compounds of formula VIII can be obtained in a manner analogous to those used to obtain compounds of formulae II-V from the compounds of formula I.
As mentioned previously, natriuretic compounds which comprise the medicamnets of the instant invention can be modified by formation of esters, amides, etc. Esterification can be carried out, for example, by reaction with a solution of a diazoalkane, or with an anhydride or an acyl chloride. Amides can be formed by reaction with ammonia or an amine.
Natriuretic compounds of formulae II-V can be derived from the corresponding natriuretic compounds produced by the foregoing method, for example, by oxidation. In a preferred embodiment of this process, when R4xe2x95x90H, R5 is not CH3.
A preferred oxidizer for the foregoing method is a solution of ferric chloride. Other oxidants, such as KMnO4, SeO2, CrO3, H2O2, m-chloroperbenzoic acid, Caro acid, OsO4, HIO4, potassium ferricyanide, silver chromate or sodium perborate, can also be used.
Scheme 1 illustrates the relationship between exemplary compounds of formulae I-V. Note that Scheme 1 depicts the relationships between the S-enantiomers. The same relationships exist between the corresponding R-enantiomers. A wide variety of natriuretic compounds within the scope of the instant invention can be obtained in the manner illustrated. 
Formulations of medicaments comprising xcex3-tocopherol and LLU-xcex1, (S)-LLU-xcex1, or other xcex3-tocopherol derivatives, detailed above, are as follows. Racemic LLU-xcex1 is synthesized or isolated and may be present as the free acid or a simple ester. Racemic LLU-xcex1 is added to the differing concentrations of xcex3-tocopherol with or without a suitable filler. A supplement comprising xcex3-tocopherol and racemic LLU-xcex1 preferably contains 5% to 95% (weight to weight) xcex3-tocopherol mixed with 5% to 95% racemic LLU-xcex1, and may also include other tocopherols. More preferably, the compositions of this embodiment of the invention include between 25% and 60% racemic LLU-xcex1, or still more preferably no more than 50% (weight to weight) racemic LLU-xcex1. A particularly preferred composition includes 26% (weight to weight) racemic LLU-xcex1 with the remaining amount of the supplement being composed of tocopherols and a suitable filler, with at least 65% of the tocopherols being xcex3-tocopherol.
Soft gelatin capsules comprising commercially available xcex3-tocopherol are fortified with (S)-LLU-xcex1 using the same compositions, above. (S)-LLU-xcex1 is synthesized or isolated, as detailed above or in the following examples, and may be present as the free acid or a simple ester. (S)-LLU-xcex1 is added to the formulations of the xcex3-tocopherol supplements mentioned above with or without a suitable filler. A supplement comprising xcex3-tocopherol and (S)-LLU-xcex1 preferably contains 5% to 95% (weight to weight) xcex3-tocopherol mixed with 5% to 95% (S)-LLU-xcex1, and may also include other tocopherols. More preferably, the compositions of this embodiment of the invention include between 25% and 60% (S)-LLU-xcex1, or still more preferably no more than 50% (weight to weight) (S)-LLU-xcex1. A particularly preferred composition includes 26% (weight to weight) (S)-LLU-xcex1 with the remaining amount of the supplement being composed of tocopherols and a suitable filler, with at least 65% of the tocopherols being xcex3-tocopherol.
Alternatively, soft gelatin capsules comprising commercially available xcex3-tocopherol are fortified with a xcex3-tocopherol derivative. The xcex3-tocopherol derivative is synthesized or isolated, as detailed above or in the following examples, and may be present as the free acid or a simple ester. An xcex3-tocopherol derivative is added to the formulations of the xcex3-tocopherol supplements mentioned above with or without a suitable filler. A supplement comprising xcex3-tocopherol and a xcex3-tocopherol derivative preferably contains 5% to 95% (weight to weight) xcex3-tocopherol mixed with 5% to 95% xcex3-tocopherol derivative, and may also include other tocopherols. More preferably, the compositions of this embodiment of the invention include between 25% and 60% xcex3-tocopherol derivative, or still more preferably no more than 50% (weight to weight) xcex3-tocopherol derivative. A particularly preferred composition includes 26% (weight to weight) xcex3-tocopherol derivative with the remaining amount of the supplement being composed of tocopherols and a suitable filler, with at least 65% of the tocopherols being xcex3-tocopherol. Other tocopherols can be included in the formulations, including (xcex1-tocopherol, xcex2-tocopherol and xcex4-tocopherol. In certain circumstances, xcex4-tocopherol can substitute for xcex3-tocopherol in the formulations and methods described herein.
The preferred method of administering principally xcex3-tocopherol or the formulation comprising xcex3-tocopherol and racemic LLU-xcex1, (S)-LLU-xcex1, or xcex3-tocopherol derivative is orally via soft gelatin capsules, however, several methods of administering these therapeutics would be within the skill of one in the art. Gamma-tocopherol or the formulations mentioned above can be administered neat, as mixtures with other physiologically acceptable active or inactive materials such as moistening agents, flavoring agents, binding agents, and extenders, as well as other compounds having pharmacological activities, such as other diuretics which increase the distal delivery of sodium, other anti-cancer therapeutics, other high blood pressure medicaments, other anti-hypertensive agents, or other mixtures of tocopherols. It may also be administered with physiologically suitable carriers such as, for example, olive oil, sesame oil, or other lipid. The compounds can be administered orally or parenterally, for example, by injection. Injection can be subcutaneous, intravenous, or by intramuscular injection.
The total daily dose of 200-800 mg can consist of a single individual dose or multiple doses given at intervals. Dosages within these ranges can also be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits have been obtained. Amounts of the compounds described herein which are therapeutically effective against specific diseases can also be determined through routine investigation.
The following examples are intended to illustrate, but not limit the invention. While they are typical of those that might be used, other procedures known to those skilled in the art may be alternatively employed. In the examples, the following abbreviations are used:
EI electron impact
FR furosemide response
FT-IR Fourier-transform infrared spectroscopy
HPLC high performance liquid chromatography
MAP mean arterial pressure
MDBK Madin-Darby bovine kidney
MS mass spectrometry
NMR nuclear magnetic resonance
PBS phosphate buffered saline
Rn natriuretic ratio
RP-HPLC reverse-phase high performance liquid chromatography
SR sample response
UNaV urine concentration of sodium X urine volume per time